Micellar systems

ABSTRACT

A complex is described that is deliverable to a cell comprising inserting a nucleic acid or other cargo into a reverse micelle. The reverse micelle has the property to compact the nucleic acid for easier delivery.

[0001] The following specification is a Continuation-In-Part of U.S.patent application Ser. No. 09/354,957 filed on Jul. 16, 1999.

FEDERALLY SPONSORED RESEARCH

[0002] N/A

FIELD OF THE INVENTION

[0003] The invention generally relates to micellar systems for use inbiologic systems. More particularly, a process is provided for the useof reverse micelles for the delivery of nucleic acids and genes tocells.

BACKGROUND

[0004] Biologically active compounds such as proteins, enzymes, andnucleic acids have been delivered to the cells using amphipathiccompounds that contain both hydrophobic and hydrophilic domains.Typically these amphipathic compounds are organized into vesicularstructures such as liposomes, micellar, or inverse micellar structures.Liposomes can contain an aqueous volume that is entirely enclosed by amembrane composed of lipid molecules (usually phospholipids) (R.C. New,p. 1, chapter 1, “Introduction” in Liposomes: A Practical Approach, ed.R.C. New IRL Press at Oxford University Press, Oxford, 1990). Micellesand inverse micelles are microscopic vesicles that contain amphipathicmolecules but do not contain an aqueous volume that is entirely enclosedby a membrane. In micelles the hydrophilic part of the amphipathiccompound is on the outside (on the surface of the vesicle) whereas ininverse micelles the hydrophobic part of the amphipathic compound is onthe outside. The inverse micelles thus contain a polar core that cansolubilize both water and macromolecules within the inverse micelle. Asthe volume of the core aqueous pool increases the aqueous environmentbegins to match the physical and chemical characteristics of bulk water.The resulting inverse micelle can be referred to as a microemulsion ofwater in oil (Schelly, Z. A. Current Opinion in Colloid and InterfaceScience, 37-41, 1997; Castro, M. J. M., Cabral, J. M. S. Biotech. Adv.6, 151-167, 1988).

[0005] Microemulsions are isotropic, thermodynamically stable solutionsin which substantial amounts of two immiscible liquids (water and oil)are brought into a single phase due to a surfactant or mixture ofsurfactants. The spontaneously formed colloidal particles are globulardroplets of the minor solvent, surrounded by a monolayer of surfactantmolecules. The spontaneous curvature, HO of the surfactant monolayer atthe oil/water interface dictates the phase behavior and microstructureof the vesicle. Hydrophilic surfactants produce oil in water (O/W)microemulsions (H0>0), whereas lipophilic surfactants produce water inoil (W/O) microemulsions. When the hydrophile-lipophile properties ofthe surfactant monolayer at the water/oil interface are balancedbicontinuous-type microemulsions are formed (H0=0).

[0006] Positively-charged, neutral, and negatively-charged liposomeshave been used to deliver nucleic acids to cells. For example, plasmidDNA expression in the liver has been achieved via liposomes delivered bytail vein or intraportal routes. Positively-charged micelles have alsobeen used to package nucleic acids into complexes for the delivery ofthe nucleic acid to cells. Negatively-charged micelles have been used tocondense DNA, however they have not been used for the delivery ofnucleic acids to cells (Imre, V. E., Luisi, P. L. Biochemical andBiophysical Research Communications, 107, 538-545, 1982). This isbecause the previous efforts relied upon the positive-charge of themicelles to provide a cross-bridge between the polyanionic nucleic acidsand the polyanionic surfaces of the cells. Micelles that are notpositively-charged, or that do not form a positively charged complexcannot perform this function. For example, a recent report demonstratedthe use of a cationic detergent to compact DNA, resulting in theformation of a stable, negatively-charged particle (Blessing, T., Remy,J. S., Behr, J. P. Proc. Natl. Acad. Sci. USA, 95, 1427-1431, 1998). Acationic detergent containing a free thiol was utilized which allowedfor an oxidative dimerization of the surfactant to the disulfide in thepresence of DNA. However, as expected, the negatively-charged complexwas not effective for transfection. Reverse (water in oil) micelles hasalso been used to make cell-like compartments for molecular evolution ofnucleic acids (Tawfik, D. S. and Griffiths, A.D. Nature Biotechnology16:652, 1998). Cleavable micellar systems was not used in this system.

[0007] In addition, Wolff et al. have developed a method for thepreparation of DNA/amphipathic complexes including micelles in which atleast one amphipathic compound layer that surrounds a non-aqueous corethat contains a polyion such as a nucleic acid (Wolff, J., Budker, V.,and Gurevich, V. U.S. Pat. No. 5,635,487).

[0008] Cleavable Micelles

[0009] A new area in micelle technology involves the use of cleavablesurfactants to form the micelle. Surfactants containing an acetallinkage, azo-containing surfactants, elimination of an ammonium salt,quaternary hydrazonium surfactants, 2-alkoxy-N,N-dimethylamine N-oxides,and ester containing surfactants such as ester containing quaternaryammonium compounds and esters containing a sugar have been developed.

[0010] These cleavable surfactants within micelles are designed todecompose on exposure to strong acid, ultraviolet light, alkali, andheat. These conditions are very harsh and are not compatible withretention of biologic activity of biologic compounds such as proteins ornucleic acids. Thus, biologically active compounds have not beenpurified using reverse micelles containing cleavable surfactants.

[0011] Complexation of Nucleic Acids with Polycations

[0012] Polymers are used for drug delivery for a variety of therapeuticpurposes. Polymers have also been used for the delivery of nucleic acids(polynucleotides and oligonucleotides) to cells for therapeutic purposesthat have been termed gene therapy or anti-sense therapy. One of theseveral methods of nucleic acid delivery to the cells is the use ofDNA-polycation complexes. It was shown that cationic proteins likehistones and protamines or synthetic polymers like polylysine,polyarginine, polyornithine, DEAE dextran, polybrene, andpolyethylenimine were effective intracellular delivery agents whilesmall polycations like spermine were ineffective. As a result the mainmechanism of DNA translocation to the intracellular space might benon-specific adsorptive endocytosis which may be more effective thenliquid endocytosis or receptor-mediated endocytosis. Furthermore,polycations are a very convenient linker for attaching specificreceptors to DNA and as result, DNA-polycation complexes can be targetedto specific cell types.

[0013] There are a variety of molecules (gene transfer enhancingsignals) that can be covalently attached to the gene in order to enableor enhance its cellular transport. These include signals that enhancecellular binding to receptors, cytoplasmic transport to the nucleus andnuclear entry or release from endosomes or other intracellular vesicles.

[0014] Nuclear localizing signals enhance the entry of the gene into thenucleus or directs the gene into the proximity of the nucleus. Suchnuclear transport signals can be a protein or a peptide such as the SV40large T ag NLS or the nucleoplasmin NLS.

[0015] Other molecules include ligands that bind to cellular receptorson the membrane surface increasing contact of the gene with the cell.These can include targeting group such as agents that target to theasialoglycoprotein receptor by using asiologlycoproteins or galactoseresidues. Other proteins such as insulin, EGF, or transferrin can beused for targeting. Peptides that include the RGD sequence can be usedto target many cells. Chemical groups that react with sulfhydryl ordisulfide groups on cells can also be used to target many types ofcells. Folate and other vitamins can also be used for targeting. Othertargeting groups include molecules that interact with membranes such asfatty acids, cholesterol, dansyl compounds, and amphotericinderivatives.

[0016] Size of a DNA complex may be a factor for gene delivery in vivo.Many times, the size of DNA that is of interest is large, and one methodof delivery utilizes compaction techniques. The DNA complex needs tocross the endothelial barrier and reach the parenchymal cells ofinterest. The largest endothelia fenestrae (holes in the endothelialbarrier) occur in the liver and have average diameter of 100 nm. Thetrans-epithelial pores in other organs are much smaller, for example,muscle endothelium can be described as a structure which has a largenumber of small pores with a radius of 4 nm, and a very low number oflarge pores with a radius of 20-30 nm.(Rippe, B. Physiological Rev,1994). The size of the DNA complex is also important for the cellularuptake process. After binding to the target cells the DNA complex shouldbe taken up by endocytosis. Since the endocytic vesicles have ahomogenous internal diameter of about 100 nm in hepatocytes, and are ofsimilar size in other cell types, the DNA is compacted to be smallerthan 100 nm.

[0017] Compaction of DNA

[0018] There are two major approaches for compacting DNA: 1. Multivalentcations with a charge of three or higher have been shown to condenseDNA. These include spermidine, spennine, Co(NH3)63+,Fe3+, and natural orsynthetic polymers such as histone H1, protamine, polylysine, andpolyethylenimine. One analysis has shown DNA condensation to be favoredwhen 90% or more of the charges along the sugar-phosphate backbone areneutralized (Wilson, R. W., Bloomfield, V. A. Biochemistry 18,2192-2196, 1979). 2. Polymers (neutral or anionic) which can increaserepulsion between DNA and its surroundings have been shown to compactDNA. Most significantly, spontaneous DNA self-assembly and aggregationprocess have been shown to result from the confinement of large amountsof DNA, due to excluded volume effect (Strzelecka, T. E., Rill, R. L.Biopolymers 30, 803-14, 1990; Strzelecka, T. E., Rill, R. L. Biopolymers30, 57-71, 1990). Since self-assembly is associated with locally ormacroscopically crowded DNA solutions, it is expected, that DNAinsertion into small water cavities with a size comparable to the DNAwill tend to form mono or oligomolecular compact structures.

[0019] Micelles and Reverse Micelles

[0020] Reverse micelles (water in oil microemulsions) are widely used asa host for biomolecules. Examples exist of both recovery ofextracellular proteins from a culture broth and recovery ofintracellular proteins. Although widely used, recovery of thebiomolecules is difficult due to the stability of the formed micelle anddue to incomplete recovery during the extraction process. Similarly,purification of DNA or other biomolecules from endotoxin and plasma isdifficult to accomplish. One common method employing Triton results inincomplete separation of the DNA or biomolecules from the emulsion.

[0021] Reverse micelles have been widely used as a host for enzymaticreactions to take place. In many examples, enzymatic activity has beenshown to increase with micelles, and has allowed enzymatic reactions tobe conducted on water insoluble substrates. Additionally, enzymaticactivity of whole cells entrapped in reverse micelles has beeninvestigated (Gajjar, L., Singh, A., Dubey, R. S., Srivastava, R. C.Applied Biochemistry and Biotechnology, 66, 159-172, 1997). The cationicsurfactant cetyl pyridinuim chloride was utilized to entrap Baker'syeast and Brewer's yeast inside a reverse micelle.

[0022] Micelles have also been used as a reaction media. For example, amicelle has been used to study the kinetic and synthetic applications ofthe dehydrobromination of 2-(p-nitrophenyl) ethyl bromide. Additionally,micelles have found use as an emulsifier for emulsion polymerizations.

[0023] Micelles have been utilized for drug delivery. For example, an ABblock copolymer has been investigated for the micellar delivery ofhydrophobic drugs. Transport and metabolism of thymidine analogues hasbeen investigated via intestinal absorption utilizing a micellarsolution of sodium glycocholate. Additionally, several examples ofmicelle use in transdermal applications have appeared. For example,sucrose laurate has been utilized for topical preparations ofcyclosporin A.

SUMMARY

[0024] The present invention provides for the transfer ofpolynucleotides, and biologically active compounds into parenchymalcells within tissues in situ and in vivo, utilizing reverse micellesdelivered intravasculary, intrarterially, intravenous, orally,intraduodenaly, via the jejunum (or ileum or colon), rectally,transdermally, subcutaneously, intramuscularly, intraperitoneally,intraparenterally, via direct. injections into tissues such as theliver, lung, heart, muscle, spleen, pancreas, brain (includingintraventricular), spinal cord, ganglion, lymph nodes, lymphatic system,adipose tissues, thyroid tissue, adrenal glands, kidneys, prostate,blood cells, bone marrow cells, cancer cells, tumors, eye retina, viathe bile duct, or via mucosal membranes such as in the mouth, nose,throat, vagina or rectum or into ducts of the salivary or other exocrineglands.

[0025] By “delivered” we mean that the polynucleotide becomes associatedwith the cell. The polynucleotide can be on the membrane of the cell orinside the cytoplasm, nucleus, or other organelle of the cell. Theprocess of delivering a polynucleotide to a cell has also been commonlytermed “transfection” or the process of “transfecting” and also it hasbeen termed “transformation”. The polynucleotide could be used toproduce a change in a cell that can be therapeutic. The delivery ofpolynucleotides or genetic material for therapeutic purposes is commonlycalled “gene therapy”. The polynucleotides or genetic material beingdelivered are generally mixed with transfection reagents prior todelivery. A biologically active compound is a compound having thepotential to react with biological components. Pharmaceuticals,proteins, peptides, hormones, cytokines, antigens and nucleic acids areexamples of biologically active compounds. The reverse micelle may benegatively-charged, zwitterionic, or neutral. Additionally, the presentinvention provides for the purification of biomolecules by solubilizingthe biomolecule into a cleavable reverse micelle and then cleaving themicelle under conditions that will not destroy the biological activityof the biomolecule. These processes can be used for transferring nucleicacids or biomolecules into cells or an organism such as for drugdelivery, or may also be used for analytical methods.

[0026] The process of utilizing cleavable reverse micelles for thepurification of biomolecules has advantages over current methodology.Isolation of the biomolecule will be enhanced by cleaving the reversemicelle. This will separate the polar group from the non-polar group onthe surfactant and therefore eliminate the formation of emulsions andtherefore simplify the isolation process. Cleavage conditions will besuch that the biological activity of the biomolecule is not destroyed.Another advantage of the invention is the use of reverse micelles forgene delivery. The reverse micelle can compact the polynucleotide, acritical step for gene delivery, especially in vivo. The micellecontaining the compacted polynucleotide can then be utilized as areaction vesicle in which additional compounds can be added to the DNA.For example, a polycation could be added to the polynucleotide/reversemicelle solution to form a polycation/polynucleotide complex within thereverse micelle. Additionally, the polynucleotide/reverse micelle systemis used as a vesicle for template polymerization of the DNA or caging ofthe DNA in which the polycation is crosslinked. A variety of chemicalreactions can take place with in the vesicle preferably withoutmodifying the nucleic acid. The polynucleotide/reverse micelle systemalso has advantages in that the micelle may be cleaved underphysiological conditions involved along the “transfection pathway.” Thesurfactant can be altered so that micellular cleavage occurs atdifferent point along this pathway. By “transfection pathway” we meanany point at which the polynucleotide/reverse micelle system isintroduced to a solution (i.e., blood, serum) that contains parenchymalcells, or to the cells (for example the skin or mucousal membranes)through the inclusion of the polynucleotide into the nucleus of theparenchymal cell.

[0027] In a preferred embodiment, described is a complex for delivery toa cell, comprising: inserting a nucleic acid into a reverse micelle. Acompacting agent may be added to the complex as well as a deliveryenhancing ligand or compound.

[0028] In a preferred embodiment, a process for delivering a complex toa cell is described, comprising inserting a nucleic acid into a reversemicelle.

[0029] In a preferred embodiment, the nucleic acid or biomolecule issolubilized into a reverse micelle with an internal water volume fordelivery of the biomolecule to parenchymal cells. A compound can beadded to the nucleic acid/micelle mixture. Such compounds includepolymers such as polyions (polycations such as spermnine, polyamines,polylysine, polyethylimine (PEI), and polyanions), proteins, peptides,enzymes, hydrophobic compounds, and amphipathic compounds (to form asecond layer around the micelle). Such compounds include compounds thatcompact the DNA, provide a cell transfer enhancing ligand or provideanother layer to the micelle.

[0030] In another preferred embodiment, the nucleic acid or biomoleculeis complexed with another molecule prior to micelle formation. Forexample, a polymer such as polylysine is added to the nucleic acid andthen crosslinked to cage the nucleic acid. When a nucleic acid is cageda polymer is formed since the polylysine (or any type of polymer) actsas a monomer that is being included in another polymer.

[0031] Another preferred embodiment provides a method of making acompound for delivery to a cell, comprising: adding one or morecompounds to the nucleic acid or biomolecule/reverse micelle complexprior to delivery to the cell, thereby providing a deliverable complex.For example, another surfactant or a polyion might be added to thecomplex. The cell can be a prokaryote or eukaryote and can be a plant,animal or mammalian cell.

[0032] Another preferred embodiment provides a method of making acompound for delivery to a cell, comprising: modifying a molecule in thepresence of the biomolecule/reverse micelle complex thereby providing adeliverable complex.

[0033] In another preferred embodiment, the parenchymal cell issolubilized within a reverse micelle. A reverse micelle containing apolynucleotide would be added to the parenchymal cell containing reversemicelle. After an appropriate amount of time, the parenchymal cell wouldbe purified, and delivered to a mammal.

[0034] In another preferred embodiment the biomolecule is solubilizedutilizing one of the following procedures.

[0035] Procedure 1

[0036] a) mixing of the biomolecule into an aqueous solution

[0037] b) then mixing the aqueous solution containing the biomoleculewith a hydrocarbon or halohydrocarbon containing a surfactant withagitation or sonication

[0038] Procedure 2

[0039] Mixing of biomolecule into a solution containing a reversemicelle with agitation or sonication.

[0040] Procedure 3

[0041] a) mixing the biomolecule into an aqueous solution

[0042] b) then extracting the aqueous solution containing thebiomolecule with a hydrocarbon or halohydrocarbon containing a reversemicelle and separating the phases.

[0043] In another preferred embodiment, the biomolecule is purifiedcomprising a step in which a reverse micelle is destroyed.

[0044] In another preferred embodiment, the biomolecule can be purifiedby utilizing one of the following procedures.

[0045] Procedure 1

[0046] a) mixing of the biomolecule into an aqueous solution

[0047] b) then mixing the aqueous solution containing the biomoleculewith a hydrocarbon or halohydrocarbon containing a surfactant withagitation or sonication

[0048] c) cleaving the reverse micelle

[0049] d) extract the biomolecule

[0050] Procedure 2

[0051] a) mixing of biomolecule into a solution containing a reversemicelle with agitation or sonication.

[0052] b) cleaving the reverse micelle

[0053] c) extract the biomolecule

[0054] Procedure 3

[0055] a) mixing the biomolecule into an aqueous solution

[0056] b) then extracting the aqueous solution containing thebiomolecule with a hydrocarbon or halohydrocarbon containing a reversemicelle and separating the phases.

[0057] c) cleaving the reverse micelle

[0058] d) extract the biomolecule

[0059] In another preferred embodiment, the surfactant is a disulfide ofgeneral formula A-S-S-B, which contains a hydrophobic group (A) and ahydrophilic group (B).

[0060] In another preferred embodiment, the surfactant can be cleaved inthe presence of the nucleic acid or biomolecule under conditions thatwill not destroy the biological activity of the nucleic acid orbiomolecule.

[0061] In another preferred embodiment, the surfactant could bechemically modified in the presence of the nucleic acid or biomolecule.For example, the surfactant can be polymerized after micelle formationto form a “shell” or cage around the nucleic acid. The surfactant couldbe cleaved separating the bulk of its hydrophilic and hydrophobic partsthus destroying its ability to act as a surfactant.

[0062] In yet another preferred embodiment, the possible surfactants canbe neutral, negatively charged, or zwitterionic but not positivelycharged. Neutral surfactants include (but not restricted to)polyoxyethylene alcohol's, polyoxyethylene isoalcohol, polyoxyethylenep-t-octyl phenol (Triton), polyoxyethylene nonylphenol, polyoxyehtyleneesters of fatty acids, polyoxyethylene sorbitol esters (Tween) andlipids. Negatively charged surfactants include (but not restricted to)di-(2-ethyl-hexyl) sodium sulfosuccinate (AOT), sodium dodecylsuphate(SDS), sodium dodecylsuphonate, and sodium dodecyl-N-sarcosinate. Thezwitterionic surfactant could contain anionic and cationic groups on thealpha and omega positions of a long aliphatic chain. For zwitterionicsurfactants that contain both anionic and cationic groups on the alphaand omega positions of a long aliphatic chain, complex formation shouldbe done under acidic conditions so that the surfactant can have apositive charge that will interact with the nucleic acid. The anionicportion is neutralized by being protonated and therefore interacts withthe non-aqueous phase. After formation of the complexes, the complexesare extracted into an aqueous solution containing a higher pH than thepH used to form the complexes.

DETAILED DESCRIPTION

[0063] A complex is described that is deliverable to a cell comprisinginserting a nucleic acid or other cargo into a reverse micelle. Thereverse micelle has the property to compact the nucleic acid for easierdelivery. The term deliverable means that the complex is capable ofbeing delivered as defined in this specification.

[0064] A process for forming a negatively-charged, zwitterionic, orneutral complex for delivery to a cell, comprising forming a cationicreverse micelle using amphipathic molecules. Then inserting abiologically active compound into the cationic reverse micelle.

[0065] Subsequently changing the charge of the cationic reverse micelleto a negatively-charged, zwitterionic, or neutral reverse micelle anddelivering it to the cell.

[0066] The amphipathic molecule may contain a silicon—heteroatom bond. Aheteroatom is any atom other than carbon or hydrogen. Examples of aheteroatom include oxygen, nitrogen, phosphorous and sulfer.

[0067] A biologically active compound is purified is when it is isolatedfrom a mixture of other compounds. It is purified when its purity isincreased where purity is defined as the percentage of the mixturecontaining the desired biologically active compound. Purification canalso indicate a process where the purity of the desired compound is notincreased but the other compounds within the mixture are changed.Purification also indicates the extraction of a biologic compound fromone mixture or solution to another mixture or solution. This can includea process where the desired biologically active compound is moved fromone solvent to another (also called extraction) or is solubilized withina solvent. A biologically active compound is a compound having thepotential to react with biological components for gene therapy purposes.Pharmaceuticals, proteins, peptides, viruses, antigens, carbohydrates(and conjugates), lipids, sacharides, oligonucleotides, and nucleicacids are examples of biologically active compounds.

[0068] A surfactant refers to a compound that contains a polar group(hydrophilic) and a non-polar (hydrophobic) group on the same molecule.Cleavable surfactant refers to a surfactant in which the polar group maybe separated from the nonpolar group by the removal of a chemical bondlocated between the two groups, or to a surfactant in which the polar ornon-polar group or both may be chemically modified such that thedetergent properties of the surfactant are destroyed. Cleavable alsomeans that the surfactant is labile (able to be destroyed or that thedetergent properties of the surfactant are able to be destroyed) andthat its surfactant properties could be destroyed by other chemicalprocesses than bond cleavage. A disulfide bond that is labile underphysiological conditions means the disulfide bond is cleaved morerapidly than oxidized glutathione or any disulfide constructed fromthiols in which one of the constituent thiols is more acidic, lower pKa,than glutathione or is activated by intramolecular attack by a freethiol. Constituent in this case means the thiols that are bondedtogether in the disulfide bond. The surfactant properties of a compoundcan be destroyed by chemical modification such as converting the polargroup into a less polar group. This can be accomplished by a number ofchemical modifications including (but not limited to) acylation,alkylation, elimination, reduction or oxidation, of an amine (or itssalt), alcohol, diol (di-alcohol) or carboxylic acid groups, or by amultistep process in which several chemical modifications are conducted(for example oxidation of an alcohol to an aldehyde (ketone) followed bynucleophilic addition to the aldehyde (ketone) resulting in an alcoholfollowed by elimination of the alcohol (or a derivative of it). Reverse(inverse) micelle refers to a surfactant with an internal aqueous pool.By “aqueous” we mean containing water, but can include buffers andsalts. Non-aqueous solutions are made up of organic solvents such ashydrocarbon or halohydrocarbon. Buffers are made from a weak acid orweak base and their salts. Buffer solutions resist changes in pH whenadditional acid or base is added to the solution. Salts are ioniccompounds that dissociate into cations and anions when dissolved insolution. Salts increase the ionic strength of a solution, andconsequently decrease interactions between nucleic acids with othercations.

[0069] A reverse micelle is destroyed when the micelle no longer existsand the monophase no longer exists. A reverse micelle is destroyed whenthe micelle is disrupted. In a preferred embodiment the reverse micelleis destroyed by chemically modifying the surfactant so that water in oilemulsion is destroyed and the phases separate. A destructible reversemicelle is a reverse micelle that can be destroyed such that the waterin oil emulsion is destroyed and the phases separate. A destructiblereverse micelle can undergo a biological, chemical, or biochemicalreaction such that the reverse micelle is destroyed. Biological,chemical, or biochemical reactions involve the formation or cleavage ofionic and/or covalent bonds. In a preferred embodiment the destructiblereverse micelle contains a surfactant that is cleavable, destroyable, orchemically modifiable. The surfactant can be a disulfide of the generalformula A-S-S- B, in which chemical group A is a hydrophobic group andchemical group B is a hydrophilic group.

[0070] The present invention also relates to a method in which abiologically active compound is delivered to a cell comprising a step inwhich the biologically active compound is mixed with abiologically-labile surfactant. A biologically-labile surfactant is asurfactant in which the hydrophobic moiety is cleaved from thehydrophilic moiety by cellular processes or its surfactant propertiesare rendered inactive within the cell, tissue or organism. Examplesinclude surfactants that contain disulfide bonds that are labile withinthe cell, tissue, or organism.

[0071] A transfection reagent is a compound or compounds used in theprior art that bind(s) to or complex(es) with polynucleotides andmediates their entry into cells. The. transfection reagent also mediatesthe binding and internalization of polynucleotides into cells. Examplesof transfection reagents include cationic liposomes and lipids, calciumphosphate precipitates, and polylysine complexes. At times, thetransfection reagent has a net positive charge that binds to thepolynucleotide's negative charge. The transfection reagent mediatesbinding of polynucleotides to cell via its positive charge (that bindsto the cell membrane's negative charge) or via ligands that bind toreceptors in the cell. For example, cationic liposomes or polylysinecomplexes have net positive charges that enable them to bind to DNA.

[0072] Other vehicles are also used, in the prior art, to transfer genesinto cells. These include complexing the polynucleotides on particlesthat are then accelerated into the cell. This is termed biolistic or guntechniques. Other methods include eletroporation in which a device isused to give an electric charge to cells. The charge increases thepermeability of the cell.

[0073] The term “nucleic acid” is a term of art that refers to a polymercontaining at least two nucleotides. “Nucleotides” contain a sugardeoxyribose (in DNA) or ribose (in RNA), a base, and a phosphate group.Nucleotides are linked together through the phosphate groups. “Bases”include purines and pyrimidines, which further include natural compoundsadenine, thymine, guanine, cytosine, uracil, inosine, and syntheticderivatives of purines and pyrimidines, or natural analogs. Nucleotidesare the monomeric units of nucleic acid polymers. A “polynucleotide” isdistinguished here from an “oligonucleotide” by containing more than 80monomeric units; oligonucleotides contain from 2 to 80 nucleotides. Theterm nucleic acid includes deoxyribonucleic acid (“DNA”) and ribonucleicacid (“RNA”). DNA may be in the form of anti-sense, plasmid DNA, partsof a plasmid DNA, vectors (P1, PAC, BAC, YAC, artificial chromosomes),expression cassettes, chimeric sequences, chromosomal DNA, orderivatives of these groups. RNA may be in the form of oligonucleotideRNA, tRNA (transfer RNA), snRNA (small nuclear RNA), rRNA (ribosomalRNA), mRNA (messenger RNA), anti-sense RNA, ribozymes, chimericsequences, or derivatives of these groups. “Anti-sense” is a nucleicacid that interferes with the function of DNA and/or RNA. This mayresult in suppression of expression. Natural nucleic acids have aphosphate backbone, artificial nucleic acids may contain other. types ofbackbones, nucleotides, or bases. These include PNAs (peptide nucleicacids), phosphothionates, and other variants of the phosphate backboneof native nucleic acids. In addition, DNA and RNA may be single, double,triple, or quadruple stranded. “Expression cassette” refers to a naturalor recombinantly produced nucleic acid which is capable of expressingprotein(s). A DNA expression cassette typically includes a promoter(allowing transcription initiation), and a sequence encoding one or moreproteins. Optionally, the expression cassette may includetranscriptional enhancers, non-coding sequences, splicing signals,transcription termination signals, and polyadenylation signals. An RNAexpression cassette typically includes a translation initiation codon(allowing translation initiation), and a sequence encoding one or moreproteins. Optionally, the expression cassette may include translationtermination signals, a polyadenosine sequence, internal ribosome entrysites (IRES), and non-coding sequences.

[0074] Delivery of a nucleic acid means to transfer a nucleic acid froma container outside a mammal to near or within the outer cell membraneof a cell in the mammal. The term “transfection” may be used, ingeneral, as a substitute for the term “delivery,” or, more specifically,the transfer of a nucleic acid from directly outside a cell membrane towithin the cell membrane. The transferred (or “transfected”) nucleicacid may contain an expression cassette. If the nucleic acid is aprimary RNA transcript that is processed into messenger RNA, a ribosometranslates the messenger RNA to produce a protein within the cytoplasm.If the nucleic acid is a DNA, it enters the nucleus where it istranscribed into a messenger RNA that is transported into the cytoplasmwhere it is translated into a protein. Therefore if a nucleic acidexpresses its cognate protein, then it must have entered a cell. Aprotein may subsequently be degraded into peptides, which may bepresented to the immune system.

[0075] Polypeptide refers to a linear series of amino acid residuesconnected to one another by peptide bonds between the alpha-amino groupand carboxyl group of contiguous amino acid residues.

[0076] “Protein” refers herein to a linear series of greater than 2amino acid residues connected one to another as in a polypeptide. A“therapeutic” effect of the protein in. attenuating or preventing thedisease state can be accomplished by the protein either staying withinthe cell, remaining attached to the cell in the membrane, or beingsecreted and dissociated from the cell where it can enter the generalcirculation and blood. Secreted proteins that can be therapeutic includehormones, cytokines, growth factors, clotting factors, anti-proteaseproteins (e.g., alphal-antitrypsin), angiogenie proteins (e.g., vascularendothelial growth factor, fibroblast growth factors), anti-angiogenicproteins (e.g., endostatin, angiostatin), and other proteins that arepresent in the blood. Proteins on the membrane can have a therapeuticeffect by providing a receptor for the cell to take up a protein orlipoprotein (e.g., low density lipoprotein receptor). Therapeuticproteins that stay within the cell (“intracellular proteins”) can beenzymes that clear a circulating toxic metabolite as in phenylketonuria.They can also cause a cancer cell to be less proliferative or cancerous(e.g., less metastatic), or interfere with the replication of a virus.Intracellular proteins can be part of the cytoskeleton (e.g., actin,dystrophin, myosins, sarcoglycans, dystroglycans) and thus have atherapeutic effect in cardiomyopathies and musculoskeletal diseases(e.g., Duchenne muscular dystrophy, limb-girdle disease). Othertherapeutic proteins of particular interest to treating heart diseaseinclude polypeptides affecting cardiac contractility (e.g., calcium andsodium channels), inhibitors of restenosis (e.g., nitric oxidesynthetase), angiogenic factors, and anti-angiogenic factors.

[0077] Biomolecule refers to peptides, polypeptides, proteins, enzymes,polynucleotides, oligonucleotides, viruses, antigens, carbohydrates (andcongugates), lipids, and sacharides.

[0078] Enzymes are proteins evolved by the cells of living organisms forthe specific function of catalyzing chemical reactions.

[0079] A chemical reaction can take place within the micelle and nucleicacid complex. A chemical reaction is defined as the formation orcleavage of covalent or ionic bonds. As a result of the chemicalreaction a polymer can be formed. A polymer is defined as a compoundcontaining more than two monomers. A monomer is a compound that can beattached to itself or another monomer and thus form a polymer. In onepreferred embodiment the surfactant is polymerized by chain or steppolymerization and then the. surfactant properties are destroyed. Thisdestruction of the surfactant properties could be a accomplished bybreaking a chemical bond and separating the hydrophilic and hydrophobicmoieties.

[0080] A chemical reaction can be used to attach a gene transferenhancing signal to the nucleic acid complex. The gene transferenhancing signal (or abbreviated as the Signal) is defined in thisspecification as a molecule that modifies the nucleic acid complex andcan direct it or the nucleic acid to a cell location (such as tissue) orlocation in a cell (such as the nucleus) either in culture or in a wholeorganism. By modifying the cellular or tissue location of the foreigngene, the expression of the foreign gene can be enhanced.

[0081] The gene transfer enhancing signal can be a protein, peptide,lipid, steroid, sugar, carbohydrate, (non-expressing) nucleic acid orsynthetic compound. The gene transfer enhancing signals enhance cellularbinding to receptors, cytoplasmic transport to the nucleus and nuclearentry or release from endosomes or other intracellular vesicles.

[0082] Nuclear localizing signals enhance the targeting of the gene intoproximity of the nucleus and/or its entry into the nucleus. Such nucleartransport signals can be a protein or a peptide such as the SV40 large Tag NLS or the nucleoplasmin NLS. These nuclear localizing signalsinteract with a variety of nuclear transport factors such as the NLSreceptor (karyopherin alpha) which then interacts with karyopherin beta.The nuclear transport proteins themselves could also function as NLS'ssince they are targeted to the nuclear pore and nucleus.

[0083] Signals that enhance release from intracellular compartments(releasing signals) can cause DNA release from intracellularcompartments such as endosomes (early and late), lysosomes, phagosomes,vesicle, endoplasmic reticulum, golgi apparatus, trans golgi network(TGN), and sarcoplasmic reticulum. Release includes movement out of anintracellular compartment into cytoplasm or into an organelle such asthe nucleus. Releasing signals include chemicals such as chloroquine,bafilomycin or Brefeldin A1. and the ER-retaining signal (KDELsequence), viral components such as influenza virus hemagglutininsubunit HA-2 peptides and other types of amphipathic peptides.

[0084] Cellular receptor signals are any signal that enhances theassociation of the gene with a cell. This can be accomplished by eitherincreasing the binding of the gene to the cell surface and/or itsassociation with an intracellular compartment, for example: ligands thatenhance endocytosis by enhancing binding the cell surface. This includesagents that target to the asialoglycoprotein receptor by usingasiologlycoproteins or galactose residues. Other proteins such asinsulin, EGF, or transferrin can be used for targeting. Peptides thatinclude the RGD sequence can be used to target many cells. Chemicalgroups that react with sulfhydryl or disulfide groups on cells can alsobe used to target many types of cells. Folate and other vitamins canalso be used for targeting. Other targeting groups include moleculesthat interact with membranes such as lipids fatty acids, cholesterol,dansyl compounds, and amphotericin derivatives. In addition viralproteins could be used to bind cells.

[0085] A polynucleotide can be delivered to a cell in order to produce acellular change that is therapeutic. The delivery of polynucleotides orother genetic material for therapeutic purposes (the art of improvinghealth in an animal including treatment or prevention of disease) isgene therapy. The polynucleotides are coded to express a whole orpartial protein, or may be anti-sense, and can be delivered eitherdirectly to the organism in situ or indirectly by transfer to a cellthat is then transplanted into the organism. The protein can be missingor defective in an organism as a result of genetic, inherited oracquired defect in its genome. For example, a polynucleotide may becoded to express the protein dystrophin that is missing or defective inDuchenne muscular dystrophy. The coded polynucleotide is delivered to aselected group or groups of cells and incorporated into those cell'sgenome or remain apart from the cell's genome. Subsequently, dystrophinis produced by the formerly deficient cells. Other examples of imperfectprotein production that can be treated with gene therapy include theaddition of the protein clotting factors that are missing in thehemophilia's and enzymes that are defective in inborn errors ofmetabolism such as phenylalanine hydroxylase. A delivered polynucleotidecan also be therapeutic in acquired disorders such as neurodegenerativedisorders, cancer, heart disease, and infections. The polynucleotide.has its therapeutic effect by entering the cell. Entry into the cell isrequired for the polynucleotide to produce the therapeutic protein, toblock the production of a protein, or to decrease the amount of a RNA.

[0086] A therapeutic effect of the protein in attenuating or preventingthe disease state can be accomplished by the protein either stayingwithin the cell, remaining attached to the cell in the membrane or beingsecreted and dissociating from the cell where it can enter the generalcirculation and blood. Secreted proteins that can be therapeutic includehormones, cytokines, growth factors, clotting factors, anti-proteaseproteins (e.g. alpha-antitrypsin) and other proteins that are present inthe blood. Proteins on the membrane can have a therapeutic effect byproviding a receptor for the cell to take up a protein or lipoprotein.For example, the low density lipoprotein (LDL) receptor could beexpressed in hepatocytes and lower blood cholesterol levels and therebyprevent atherosclerotic lesions that can cause strokes or myocardialinfarction. Therapeutic proteins that stay within the cell can beenzymes that clear a circulating toxic metabolite as in phenylketonuria.They can also cause a cancer cell to be less proliferative or cancerous(e.g. less metastatic). A protein within a cell could also interferewith the replication of a virus.

[0087] The delivered polynucleotide can stay within the cytoplasm ornucleus apart from the endogenous genetic material. Alternatively, thepolynucleotide could recombine (become a part of) the endogenous geneticmaterial. For example, DNA can insert into chromosomal DNA by eitherhomologous or non-homologous recombination.

[0088] Parenchymal cells are the distinguishing cells of a gland ororgan contained in and supported by the connective tissue framework. Theparenchymal cells typically perform a function that is unique to theparticular organ. The term “parenchymal” often excludes cells that arecommon to many organs and tissues such as fibroblasts and endothelialcells within the blood vessels. In a liver organ, the parenchymal cellsinclude hepatocytes, Kupffer cells and the epithelial cells that linethe biliary tract and bile ductules. The major constituent of the liverparenchyma are polyhedral hepatocytes (also known as hepatic cells) thatpresents at least one side to an hepatic sinusoid and apposed sides to abile canaliculus. Liver cells that are not parenchymal. cells includecells within the blood vessels such as the endothelial cells orfibroblast cells.

[0089] In striated muscle, the parenchymal cells include myoblasts,satellite cells, myotubules, and myofibers. In cardiac muscle, theparenchymal cells include the myocardium also known as cardiac musclefibers or cardiac muscle cells and the cells of the impulse connectingsystem such as those that constitute the sinoatrial node,atrioventricular node, and atrioventricular bundle. In a pancreas, theparenchymal cells include cells within the acini such as zymogeniccells, centroacinar cells, and basal or basket cells and cells withinthe islets of Langerhans such as alpha and beta cells. In spleen,thymus, lymph nodes and bone marrow, the parenchymal cells includereticular cells and blood cells (or precursors to blood cells) such aslymphocytes, monocytes, plasma cells and macrophages.

[0090] In the nervous system which includes the central nervous system(the brain and spinal cord) peripheral nerves, and ganglia, theparenchymal cells include neurons, glial cells, microglial cells,oligodendrocytes, Schwann cells, and epithelial cells of the choroidplexus. In the kidney, parenchymal cells include cells of collectingtubules and the proximal and distal tubular cells. In the prostate, theparenchyma includes epithelial cells. In glandular tissues and organs,the parenchymal cells include cells that produce hormones. In theparathyroid glands, the parenchymal cells include the principal cells(chief cells) and oxyphilic cells. In the thyroid gland, the parenchymalcells include follicular epithelial cells and parafollicular cells. Inthe adrenal glands, the parenchymal cells include the epithelial cellswithin the adrenal cortex and the polyhedral cells within the adrenalmedulla. In the parenchyma of the gastrointestinal tract such as theesophagus, stomach, and intestines, the parenchymal cells includeepithelial cells, glandular cells, basal, and goblet cells. In theparenchyma of lung, the parenchymal cells include the epithelial cells,mucus cells, goblet cells, and alveolar cells. In fat tissue, theparenchymal cells include adipose cells or adipocytes. In the skin, theparenchymal cells include the epithelial cells of the epidermis,melanocytes, cells of the sweat glands, and cells of the hair root. Incartilage, the parenchyma includes chondrocytes. In bone, the parenchymaincludes osteoblasts, osteocytes, and osteoclasts. Intravascular refersto an intravascular route of administration that enables a polymer,oligonucleotide, or polynucleotide to be delivered to cells more evenlydistributed and more efficiently than direct injections. Intravascularherein means within an internal tubular structure called a vessel thatis connected to a tissue or organ within the body of an animal,including mammals. Within the cavity of the tubular structure, a bodilyfluid flows to or from the body part. Examples of bodily fluid includeblood, lymphatic fluid, or bile. Examples of vessels include arteries,arterioles, capillaries, venules, sinusoids, veins, lymphatics, and bileducts. The intravascular route includes delivery through the bloodvessels such as an artery or a vein. “Intracoronary” refers to anintravascular route for delivery to the heart wherein the blood vesselsare the coronary arteries and veins.

[0091] Permeability is defined herein as the propensity formacromolecules such as nucleic acids to move through vessel walls andenter the extravascular space. One measure of permeability is the rateat which macromolecules move through the vessel wall and out of thevessel. Another measure of permeability is the lack of force thatresists the movement through the vessel wall and out of the vessel.Vessels contain elements that prevent macromolecules from leaving theintravascular space (internal cavity of the vessel). These elementsinclude endothelial cells and connective material (e.g., collagen). Highpermeability indicates that there are fewer of these elements that canblock the egress of macromolecules and that the spaces between theseelements are larger and more numerous. In this context, highpermeability enables a high percentage of nucleic acids being deliveredto leave the intravascular space, while low permeability indicates thata low percentage of the nucleic acids will leave the intravascularspace.

[0092] The permeability of a blood vessel can be increased by increasingthe intravascular hydrostatic pressure. In a preferred embodiment, theintravascular hydrostatic pressure is increased by rapidly (from 1seconds to 30 minutes) injecting a nucleic acid in solution into theblood vessel, which increases the hydrostatic pressure. In anotherpreferred embodiment, hydrostatic pressure is increased by obstructingthe outflow of the injection solution from the tissue for a period oftime sufficient to allow delivery of a nucleic acid. Obstructing meansto block or impede the outflow of injection fluid, thereby transiently(reversibly) blocking the outflow of the blood. Furthermore, rapid.injection may be combined with obstructing the outflow in yet anotherpreferred embodiment. For example, an afferent vessel supplying an organis rapidly injected while the efferent vessel draining the tissue isblocked transiently (e.g., by ligation, or by an inflated intravascularballoon). The efferent vessel (also called the venous outflow or tract)draining outflow from the tissue is partially or totally clamped for aperiod of time sufficient to allow delivery of a nucleic acid. In thereverse, an efferent vessel is injected while the corresponding afferentvessel is occluded.

[0093] An administration route involving the mucosal membranes is meantto include nasal, bronchial, inhalation into the lungs, or via the eyes.

[0094] Transdermal refers to application to mammal skin in which drugdelivery occurs by crossing the dermal layer.

[0095] Crosslinking refers to the chemical attachment of two or moremolecules with a bifunctional reagent. A bifunctional reagent is amolecule with two reactive ends. The reactive ends can be identical asin a homobifunctional molecule, or different as in a heterobifucnctionalmolecule.

[0096] Electrostatic interactions are the non-covalent association oftwo or more substances due to attractive forces between positive andnegative charges.

[0097] Amphipathic compounds have both hydrophilic (water-soluble) andhydrophobic (water-insoluble) parts.

[0098] A polycation is a polymer containing a net positive charge, forexample poly-L-lysine hydrobromide. The polycation can contain monomerunits that are charge positive, charge neutral, or charge negative,however, the net charge of the polymer must be positive. A polycationalso can mean a non-polymeric molecule which contains two or morepositive charges. A polyanion is a polymer containing a net negativecharge, for example polyglutamic acid. The polyanion can contain monomerunits that are charge negative, charge neutral, or charge positive,however, the net charge on the polymer must be negative. A polyanion canalso mean a non-polymeric molecule that contains. two or more negativecharges. The term polyion includes polycation, polyanion, zwitterionicpolymers, and neutral polymers. The term zwitterionic refers to theproduct (salt) of the reaction between an acidic group and a basic groupthat are part of the same molecule.

[0099] Mixing means the method of interdispursing two or more solvents,or solvent(s) and solute(s). Sonication and agitation are forms ofmixing. Solvent refers to a material in the liquid phase that can beused to solubilize (dissolve) a compound. Solute refers to a compounddissolved in a solvent.

[0100] Hydrocarbon means containing carbon and hydrogen atoms; andhalohydrocarbon means containing carbon, halogen (F, Cl, Br, I), andhydrogen atoms.

[0101] Alkyl means containing sp3 hybridized carbon atoms; alkenyl meanscontaining two or more sp2 hybridized carbon atoms; aklkynyl meanscontaining two or more sp hybridized carbon atoms; aralkyl meanscontaining one or more aromatic ring(s) in addition containing sp3hybridized carbon atoms; aralkenyl means containing one or more aromaticring(s) in addition to containing two or more sp2 hybridized carbonatoms; aralkynyl means containing one or more aromatic ring(s) inaddition to containing two or more sp hybridized carbon atoms; steroidincludes natural and unnatural steroids and steroid derivatives. Asteroid derivative means a sterol, a sterol in which the hydroxyl moietyhas been modified (for example, acylated), or a steroid hormone, or ananalog thereof; carbohydrates include natural and unnatural sugars (forexample glucose), and sugar derivatives (a sugar derivative means asystem in which one or more of the hydroxyl groups on the sugar moietyhas been modified (for example acylated), or a system in which one ormore of the hydroxyl groups is not present); polyoxyethylene means apolymer having two to six (n=2-6) ethylene oxide units (-(CH2CH2O)n-) ora derivative thereof; and R not identified by number is meant to be anycompatible group, for example alkyl, alkenyl, alkynyl, aralkyl,aralkenyl, or aralkynyl, and can include heteroatoms (N, O, S), andcarbonyl groups.

[0102] Hydrophilic groups indicate in qualitative terms that thechemical moiety is water-preferring. Typically, such chemical groups arewater soluble, and are hydrogen bond donors or acceptors with water.Examples of hydrophilic groups include compounds with the followingchemical moieties carbohydrates; polyoxyethylene, peptides,oligonucleotides and groups containing amines, amides, alkoxy amides,carboxylic acids, sulfurs, or hydroxyls. Hydrophobic groups indicate inqualitative terms that the chemical moiety is water-avoiding. Typically,such chemical groups are not water soluble, and tend not to hydrogenbond. Hydrocarbons are hydrophobic groups.

[0103] The terms “therapeutic” and “therapeutic results” are defined inthis application as levels of gene products, including reporter (marker)gene products, which indicate a reasonable expectation of geneexpression using similar compounds (other nucleic acids including othergenes), at levels considered sufficient by a person having ordinaryskill in the art of gene therapy. For example: Hemophilia A and B arecaused by deficiencies of the X-linked clotting factors VIII and IX,respectively. Their clinical course is greatly influenced by thepercentage of normal serum levels of factor VIII or IX: <2%, severe;2-5%, moderate; and 5-30% mild. This indicates that in severe patientsonly 2% of the normal level can be considered therapeutic. Levelsgreater than 6% prevent spontaneous bleeds but not those secondary tosurgery or injury. A person having ordinary skill in the art of genetherapy would reasonably anticipate therapeutic levels of expression ofa gene specific for a disease based upon sufficient levels of markergene results. In the Hemophilia example, if marker genes were expressedto yield a protein at a level comparable in volume to 2% of the normallevel of factor VIII, it can be reasonably expected that the gene codingfor factor VIII would also be expressed at similar levels.

[0104] There are three types of reporter (marker) gene products that areexpressed from reporter genes. The reporter gene/protein systemsinclude:

[0105] a) Intracellular gene products such as luciferase,β-galactosidase, or chloramphenicol acetyl transferase. Typically, theyare enzymes whose enzymatic activity can be easily measured.

[0106] b) Intracellular gene products such as β-galactosidase or greenfluorescent protein which identify cells expressing the reporter gene.On the basis of the intensity of cellular staining, these reporter geneproducts also yield qualitative information concerning the amount offoreign protein produced per cell.

[0107] Secreted gene products such as growth hormone, factor IX, oralphal-antitrypsin are useful for determining the amount of a secretedprotein that a gene transfer procedure can produce. The reporter geneproduct can be assayed in a small amount of blood.

EXAMPLES Compound Preparation Synthesis of (-D-Glucopyranosyl DodecaneDisulfide

[0108] To a solution of dodecane thiol (1.00 mL, 4.17 mmol, Aldrich) in20 mL CHCl3 was added sulfuryl chloride (0.74 mL, 9.18 mmol, Aldrich),and the resulting mixture was stirred at room temperature for 18 h.Removal of solvent (aspirator), afforded dodecansulfenyl chloride thatwas determined to be sufficiently pure by 1H NMR.

[0109] To a solution of dodecansulfenyl chloride (213 mg, 0.899 mmol) in2.7 mL acetonitrile was added (-D-thioglucose sodium salt (200. mg,0,917 mmol, Aldrich) and 15-crown-5 (0.18 mL, 0.899 mmol, Aldrich). Theresulting mixture was stirred at ambient temperature for 3 h, and thesolvent removed (aspirator). The residue was triturated with CHCl3 andfiltered. The residue was purified by flash column chromatography onsilica gel (0-5% MeOH in CH2Cl2). Crystallization (EtOAc) afforded 85 mg(24%) of a fine white solid.

I. Example 1 Synthesis of β-D-Glucopyranosyl Dodecane Disulfide

[0110]

[0111] To a solution of dodecane thiol (1.00 mL, 4.17 mmol, AldrichChemical Company) in 20 mL CHCl3 was added sulfuryl chloride (0.74 mL,9.18 mmol, Aldrich Chemical Company), and the resulting mixture wasstirred at room temperature for 18 h. Removal of solvent (aspirator),afforded dodecansulfenyl chloride that was determined to be sufficientlypure by 1H NMR.

[0112] To a solution of dodecansulfenyl chloride (213 mg, 0.899 mmol) in2.7 mL acetonitrile was added 1-thio-β-D-glucose sodium salt hydrate(200. mg, 0,917 mmol, Aldrich Chemical Company) and 15-crown-5 (0.18 mL,0.899 mmol, Aldrich Chemical Company). The resulting mixture was stirredat ambient temperature for 3 h, and the solvent removed (aspirator). Theresidue was triturated with CHCl3 and filtered. The residue was purifiedby flash column chromatography on silica gel (0-5% MeOH in CH2Cl2).Crystallization (EtOAc) afforded 85 mg (24%) of β-D-glucopyranosyldodecane disulfide as a fine white solid.

Example 2 Demonstration of Micelle Formation with β-D-GlucopyranosylDodecane Disulfide, and Micelle Destruction with Dithiothreitol

[0113] To a solution of β-D-Glucopyranosyl dodecane disulfide (10 mg) in1 mL CD3Cl was added 1 mL H2O. The sample was rapidly mixed resulting ina thick white emulsion. After 18 h, the organic and aqueous layers wereemulsified to approximately 95%. After 4 d, the organic and aqueouslayers remained emulsified to approximately 70%. To a 1 mL portion ofthe emulsion was added 60 μg of dithiothreitol (Aldrich ChemicalCompany), and the solution was mixed. After 30 min, the emulsion hadcleared. 5,5′-Dithiobis(2-nitrobenzioc acid) (1 mg, Aldrich ChemicalCompany) was added, resulting in a yellow solution, verifying thepresence of free sulfide. Analysis also indicated the presence ofdodecane thiol and 1-thio-β-D-glucose by TLC.

Example 3 Solubilization of PCILuc DNA in Reversed Micelles

[0114] Procedure

[0115] PCILuc DNA (pDNA) (Zhang, G., Vargo, D., Budker, V., Armstrong,N., Knechtle, S., Wolff, J. A. Human Gene Therapy, 8, 1763-1772, 1997)(11 μg) was taken up in a solution (3-67 μL) of HEPES (25 mM, pH 7.8)and EDTA (0.5 mM). Polyoxyethylene(4) lauryl ether (Brij 30) (1.2 mL,Aldrich Chemical Company) was taken up in 2,2,4-trimethylpentane (TMP)(8.8 mL, Aldrich Chemical Company). To the Brij 30/TMP solution (0.7 mL)was added the pDNA in buffer (3-67 μL). The mixtures were shaken (2 min)resulting in clear solutions. After 10 min the turbidity was determinedutilizing a fluorescence spectrophotometer (Hitachi, model F3010,extinction/emission wavelength of 529 nm). WO is defined as the molarratio of water to surfactant.

[0116] Results H2O (μL) W0 Turbidity (529 nm) 0 0 19 3 0.72 49 7 1.68 6312 2.87 63 17 4.07 82 27 6.46 2764 47 11.25 1565 67 16.04 214

[0117] Analysis

[0118] At 20° C., the pDNA solution when added to the Brij 30/TMP formsinversed micelles for an aqueous content of up to W0=4. For a solutionof Brij 30 in dodecane the hydrophile-lipophile balance (HLB)temperature has been determined to be approximately 29.2° C. (Kunieda,H. Langmuir 7,1915, 1991). For the present system it was shown thatbelow 29.2˜C., w/o microemulsion are present for a WO of less then 10.By increasing the water content, one obtains a two-phase system and thena lamellar phase. In the case of Brij 30 in TMP, a two-phase systemappears at WO of more then 6 and a lamellar phase at WO of more then 11.

Example 4 Determination of the Size of PCILuc DNA Contained in InversedMicelles

[0119] Part A. Centrifugation

[0120] Procedure

[0121] PCILuc DNA (pDNA) (Zhang, G., Vargo, D., Budker, V., Armstrong,N., Knechtle, S., Wolff, J. A. Human Gene Therapy, 8, 1763-1772, 1997)(36μg) was taken up in a solution of HEPES (25 mM, pH 7.8) and EDTA (0.5mM) (10 μL, 20 μL, 30 μL, and 50 μ). The resulting solutions were addedto a mixture of Polyoxyethylene(4) lauryl ether (Brij 30) (AldrichChemical Company)/2,2,4-trimethylpentane (TMP) (Aldrich ChemicalCompany) (1 mL, 1:7.3 v/v) and agitated. The UV adsorption wasdetermined (Perkin Elmer, UV/VIS Spectrophotometer, model Lambda 6)against 10 μL of HEPES (25 mM, pH 7.8) and EDTA (0.5 mM) buffer in Brij30/TMP (1 mL, 1:7.3 v/v). The samples were centrifuged 5 min at 15000rpm and the UV adsorption was again determined.

[0122] Results A₂₆₀ before A₂₆₀ after Conditions W0 centrifugationcentrifugation DNA in buffer — 1.07 1.07 10 μl 1.68 1.07 1.11 20 μl 3.360.99 1.14 30 μl 5.04 0.97 1.01 50 μl 8.39 2.44 ND^(a)

[0123] Analysis

[0124] At 20° C., micelles that contain pDNA (up to W0=5) are smallenough to stay in solution in the course of centrifugation. For thesesolutions, no change in the UV absorption spectra was recorded ascompared to the UV absorption of pDNA in HEPES (25 mM, pH 7.8) and EDTA(0.5 mM).

[0125] Part B. Particle Size of Micelles Without PCILuc DNA

[0126] Procedure

[0127] A solution (5 - 50 μL) of HEPES (25 mM, pH 7.8) and EDTA (0.5 mM)was added to a mixture of Brij 30/TMP (1 mL, 1:7.3 v/v) and agitated (2min). The samples were centrifuged (1 min) at 12000 rpm and the size ofmicelles measured (Particle Sizer, Brookhaven Instrument Corporation).

[0128] Results Volume of buffer (μL) W0 Size nm  0 0   1.3  5 0.84 2.910 1.68 3.4 20 3.35 5.1 30 5.04 9.7 50 8.39 indefinite

[0129] Analysis

[0130] The size of the micelles changes proportionally as the watercontent increases, from 1.3 nm for “dry” micelles to 9.7 nm for micelleswith W0=5. At a higher water content, a two-phase phase system ispresent.

[0131] Part C. Particle Size of Micelles Containing PCILuc DNA

[0132] Procedure

[0133] A solution pDNA in HEPES (25 mM, pH 7.8) and EDTA (0.5 mM) wasadded to a mixture of Brij 30/TMP (1 mL, 1:7.3 v/v) and agitated (2 min)to form micelles with a WO of 3.35. The samples were centrifuged (1 min)at 12000 rpm and the size of micelles was measured (Particle Sizer,Brookhaven Instrument Corporation).

[0134] Results DNA ng Small micelles (nm) Large micelles (nm) 0 5.1 — 404.0 16.2 80 4.7 48.7 120 4.7 62.8 160 4.4 51.7

[0135] Analysis

[0136] Two types of micelles appear to be present in the samples. Thereare small, “empty” micelles, and large pDNA containing micelles. Itappears that the size of micelles containing pDNA increases as theconcentration of pDNA increases. The micelle appears to be saturated ata size of 50-60 nm.

Example 5 Conformation of PCILuc DNA in Inverse Micelles

[0137] Procedure

[0138] pDNA (60 μg) was taken up in 10 mM potassium phosphate buffer atpH 7.5 (20 μL and 60 μL). The pDNA solutions were added to a mixture ofBrij 30/TMP (1 mL, 1:7.3 v/v) and agitated (2 min). The circulardichroism spectra were measured for each sample (cell length =0.5 cm,Spectropolarimeter 62DS, Avive Associates) at 30° C. against controlsamples prepared without the pDNA (the ellipticity value for the controlsamples were subtracted from the experimental samples).

[0139] Results

[0140] There are shifts in the position of both the positive andnegative bands and in the position of the cross-over point for the 20 μLpDNA solution (W0=3.35). Spectra that are similarly shifted are broadlydefined as -spectra, and are attributed to a condensed form of pDNA. Incontrast the spectra of the 60 μL pDNA solution (W0=10.05) resembles thespectra of DNA in buffer alone in respect to cross-over point. Howeverthis spectra is characterized by an increase in the intensity of thenegative band (maximum at 240 nm).

Example 6 PCILuc DNA Condensation

[0141] Part A. Ethidium Bromide

[0142] Procedure

[0143] A solution of pDNA in HEPES (25 mM, pH 7.8) and EDTA (0.5 mM)(3-67 μL) containing ethidium bromide (0.9 μg, Sigma Chemical Company)was added to a mixture of Brij 30/TMP (0.7 mL, 1:7.3 v/v) and agitated.After 4 h at ambient temperature, the samples were assayed utilizing afluorescence spectrophotometer (Hitachi, Model F-3010), with anexcitation wavelength of 525 nm and an emission wavelength of 595 nm.

[0144] Results Volume (μL) W0 I/Imax*100  3 0.72 15  7 1.68 13 12 2.8712 17 4.07 12.5 27 6.46 23 47 11.25 35 67 16.04 51

[0145] Analysis

[0146] The pDNA in reverse micelles of up to W0=4 is condensed.Additionally, some level of condensation is shown for micelles up toW0=16.

[0147] Part B: Determination of Rhodamine Labeled DNA Condensation in aReverse Micelle

[0148] Procedure

[0149] pDNA was modified to a level of 1 Rhodamine per 100 bases usingMirus' Label It® Rhodamine kit (Rhodamine Containing DNA LabelingReagent, Mirus Corporation). The modified pDNA (2.5 μg) was solubilizedin different volumes of HEPES (25 mM, pH 7.8) and EDTA (0.5 mM ) andadded to a solution of Brij 30/TMP (0.7 mL, 1:7.3 v/v), and agitated.The fluorescence was determined using a fluorescence spectrophotometer(Hitachi, Model F-3010), at an excitation wavelength of 591 nm, and anemission wavelength of 610 nm.

[0150] Results Buffer Volume (μL) W0 (I₆₁₀ sample/I₆₁₀ DNA inbuffer)*100  2 0.48 104   4 0.96 80  5 1.2 34 10 2.39 31 12 2.87 24 153.59 33 22 5.26 32 32 7.66 65 42 10.0 106  52 12.45 93 62 14.84 78

[0151] It should be noted that around W0=10 turbidity has significantcontribution in fluorescence. The assay indicates that under low waterconditions, pDNA does not appear to be condensed. As the amount of waterin the system is increased, the fluorescence results indicate that pDNAis condensed within the w/o microemulsion.

II. Example 7 pDNA Condensation in Reverse Micelles Procedure

[0152] pDNA was modified to a level of 1 Rhodamine per 100 bases usingstandard procedures (Label It®, Mirus Corporation). Labeled pDNA(various amounts) was taken up in HEPES (25 mM, pH 7.8) EDTA (0.5 mM )(various amounts) and was mixed with unmodified pDNA (various amounts)to afford 2.5 μg total of pDNA. The resulting solution was added to Brij30/TMP (0.7 mL, 1:7.3 v/v) and the fluorescence was determined using afluorescence spectrophotometer (Hitachi, Model F-3010), at an excitationwavelength of 591 nm, and an emission wavelength of 610 nm. Forcomparison, the fluorescence was also determined for the similar ratiosof Rh-labeled pDNA/pDNA containing 2 mM spermidine (Sigma ChemicalCompany) in HEPES (25 mM, pH 7.8) and EDTA (0.5 mM) (0.7 mL).

[0153] Results % of Fluorescence quenching % Rh-DNA W0 = 2.39 W0 = 3.59W0 = 7.18 2 mM Sp. 100  68.8 61.2 41.3 69.8 76 65.9 57.5 33.1 61 51 59  52.2 30   48 26 55.5 50.4 28.3 26.1

[0154] Analysis

[0155] The fluorescence data indicates a relatively weak affect ofRh-labeled pDNA dilution by unlabeled pDNA. On the other hand, in thesamples containing spermidine, a strong effect of the Rh-pDNA dilutionby unlabeled DNA is shown. In reverse micelles, the pDNA condensationstarts from monomolecular condensation and therefore show little effectby the dilution protocol. However, in the spermidine containing systems(non-micellular) the strong effect indicates that condensation ismultimolecular.

III. Example 8 Transmission Electron Microscope Assay

[0156] Procedure

[0157] A drop of Poly-L-lysine (PLL) (30-70KD, Sigma Chemical Company)in water (concentration of 10 mg/mL) was placed on a covered EM grid.The solution was removed, and the grid was dried. A drop of2,2,4-trimethylpentane (TMP) (Aldrich Chemical Company) in variousamounts of HEPES (25 mM, pH 7.8) and EDTA (0.5 mM) both with and withoutPCILuc DNA (PDNA) (7 μg/mL TMP) was placed on the grid.

[0158] After 5 min, the solution was removed and the grid was washedwith TMP (3×) and water (1×), and then stained with Uranyl Acetate.

[0159] Results

[0160] Samples containing 20 or 60 μL of HEPES (25 mM, pH 7.8) and EDTA(0.5 mM) in TMP (1 mL) failed to show any structures. A samplecontaining pDNA (7 μg) in HEPES (25 mM, pH 7.8) and EDTA (0.5 mM) in TMP(1 mL) also failed to show any structures. A sample containing pDNA inHEPES (25 mM, pH 7.8) and EDTA (0.5 mM) (20 μL) and TMP (1 mL)demonstrated ring like structures with an external diameter of 59.8±12.5nm and an internal diameter of 32.9±12.1 nm. A sample of pDNA in HEPES(25 mM, pH 7.8) and EDTA (0.5 mM) (60 μL) and TMP (1 mL) demonstratedlong threads with a 7-12 nm diameter. The volume of the terroid ringV=(˜2/4)(R_(out) −R_(in))² (R_(out) +R_(in)) equal 41*10³ nm³. Thevolume of “dry” PCILuc DNA is 6.4*10³ nm ³. With consideration ofpacking parameter every toroid therefore contains five pDNA's.

Example 9 PCILuc DNA/Labeled Poly-L-Lysine Interaction

[0161] Procedure

[0162] To Poly-L-lysine (PLL) (4 mg, Sigma Chemical Company) inpotassium phosphate buffer (pH 8, 0.1 mL) was added7-Chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl ) (0.4 mg, Sigma ChemicalCompany). The solution was heated at 37˜ C. for 2 h, cooled, andpurified by gel-filtration on Sephadex G-25. The fluorescence wasdetermined (Hitachi, model F-3010, excitation wavelength =466 nm,emission wavelength =540 nm), and the level of modification wasestimated to be 5%. To the NBD-PLL (5 μg) in HEPES (25 mM, pH 7.8) andEDTA (0.5 mM) (1 mL), was added pDNA, and the fluorescence was againdetermined.

[0163] Results pDNA μg 0 1 2 4 6 I₅₄₀ 41 27 21 17 16

[0164] Analysis

[0165] The interaction of the NBD-PLL with pDNA was shown tosubstantially decrease the quantum yield of fluorescence.

Example 10 PCILuc DNA/Polycation Interaction in a Reverse Micelle

[0166] Procedure

[0167] NBD-PLL (example 9) was mixed with Polyoxyethylene(4) laurylether (Brij 30) (Aldrich Chemical Company)/ 2,2,4-trimethylpentane (TMP)(Aldrich Chemical Company) (1:7.3 v/v), and then mixed with an equalvolume of Brij 30/TMP (1:7.3 v/v) that contained either HEPES (25 mM, pH7.8) and EDTA (0.5 mM) or HEPES (25 mM, pH 7.8) and EDTA (0.5 mM) withpDNA (various amounts) After 10 min at ambient temperature, thefluorescence was determined for each sample. Conditions I₅₄₀ 0.5 mL TMPDwith 5 μg NBD-PLL in 20 μL buffer + 87 0.5 mL TMPD with 20 μL buffer 0.5mL TMPD with 5 μg NBD-PLL in 20 μL buffer + 64 0.5 mL TMPD with 3.7 μgDNA in 20 μL buffer 0.5 mL TMPD with 5 μg NBD-PLL in 20 μL buffer + 380.5 mL TMPD with 11.1 μg DNA20 μL buffer

[0168] Analysis

[0169] The results from the fluorescence study indicate that pDNA inreverse micelles can interact with PLL also in reverse micelles.

IV. Example 11 PCILuc DNA/Crosslinked Polycation Interaction

[0170] Procedure

[0171] To a solution of pDNA (35 μg) in HEPES (25 mM, pH 7.8), EDTA (0.5mM), and NaCl (100 mM) (24 μL) was added Polyoxyethylene(4) lauryl ether(Brij 30) (Aldrich Chemical Company)/ 2,2,4-trimethylpentane (TMP)(Aldrich Chemical Company) (510 μL, 1:7.3 v/v). Poly-L-lysine (PLL) (95μg, Sigma Chemical Company) in HEPES (25 mM, pH 7.8), EDTA (0.5 mM), andNaCl (100 mM) (12 μL) was added to Brij 30/TMP (290 μL, 1:7.3 v/v). Theresulting solutions were mixed and heated to 40˜ C. for 30 min at whichtime Dimethyl 3,3′- dithiobispropionimidate-2HCl (DTBP, Pierce ChemicalCompany) in DMSO (various amounts of a 29.5 mg/mL solution) were added.The solution was heated to 40˜ C. for 25 min at which time HEPES (25 mM,pH 7.8), EDTA (0.5 mM), and NaCl (100 mM) (200 μL) was added, followedby EtOH (50 μL) and EtOAc (0.5 mL). After mixing and centrifugation, theaqueous layer was washed with EtOAc (2×1 mL) and Ether (2×1 mL). Thesamples were spun (5 min, 12000 rpm) and dialyzed for 16 h against HEPES(25 mM, pH 7.8) and NaCl (100 mM). The UV absorption was determined(Perkin Elmer UV/VIS Spectrophotometer, Model Lambda 6). A solution ofTO6 (Zeng, Z., Clark, S. M., Mathies, R. A., Glazer, A. N. AnalyticalBiochemistry, 252, 110-114, 1997) (2 μL, 0.5 mg/mL in water) was addedand the fluorescence was determined (Hitchi, Model F-3010, excitationwavelength=509 nm, emission wavelength=540 nm).

[0172] Results # Amount of DTBP μl % DNA recovery Fluorescence 35 μg DNA— 100  120.4 (no treatment) 1 0  3 0.275 2 3 14 1.76 3 6 19 3.07 4 12 24 4.02

[0173] Analysis

[0174] The results indicate that the pDNA-PLL complex can be partlyextracted from reverse micelles after the PLL has been crosslinked withDTBP. The pDNA in the extracted complexes is compacted because it doesnot interact with the fluorescent intercolator TO6.

Example 12 PCILuc DNA/Polyethylenimine Complexes in Reverse Micelles

[0175] Procedure

[0176] pDNA was labeled as above (Label It®, Mirus Corporation). LabeledpDNA (14 μg) was taken up in HEPES (25 mM, pH 7.8) and EDTA (0.5 mM)(various amounts) and added to Polyoxyethylene(4) lauryl ether (Brij 30)(Aldrich Chemical Company)/2,2,4-trimethylpentane (TMP) (AldrichChemical Company) (1 mL, 1:7.3 v/v). The fluorescence and turbidity ofeach sample was determined. Polyethylenimine (PEI) (30 μg, SigmaChemical Company) in HEPES (25 mM, pH 7.8) and EDTA (0.5 mM) (3 μL) wasadded to each sample. After 30 min the florescence and turbidity of eachsample was determined.

[0177] Results No PEI With PEI I₆₁₀ Turbidity I₆₁₀ Turbidity DNA inbuffer 28.45 31  8.7 76 W₀ = 0.67 14.8 105 11.5 164 1.51 9.7 103 10.2144 2.35 11.0 85 11.8 114 4.03 18.3 105 15.9 137 5.71 26.0 182 18.0 2179.06 31.6 4200 17.8 4734

[0178] Analysis

[0179] The decrease in fluorescence after the addition of PEI indicatesthat PEI was within the same micelle as the DNA and was bound to it.

Example 13 Oxidation Within a Reverse Micelle

[0180] Procedure

[0181] Cysteine Label IT® was prepared by amidation of amino Label IT®(Mirus Corporation Madison Wis.) with N-Boc-S-trityl cysteine (SigmaChemical Company) utilizing dicyclohexylcarbodiimide (Aldrich ChemicalCo.) as the coupling agent. The product was purified by precipitationwith diethyl ether. The trityl and Boc protecting groups were removedwith trifluoroacetic acid. The resulting free thiol group was protectedwith Aldrithiol-2®(Aldrich Chemical Co.) as the pyridyldithio mixeddisulfide and was purified by diethyl ether precipitation and confirmedby mass spectrometry (Sciex API 150EX).

[0182] PCILuc DNA (pDNA) (Zhang, G., Vargo, D., Budker, V., Armstrong,N., Knechtle, S., Wolff, J. A. Human Gene Therapy, 8, 1763-1772, 1997)was modified with Cysteine Label IT® at weight ratios of 0.1:1 and 0.2:1(reagent:DNA) at 37° C. for 1 hour. The labeled DNA was purified byethanol precipitation. The purified DNA was reconstituted in 20 mM MOPSpH 7.5, 0.1 mM EDTA buffer at a final concentration of 1 μg/μL. Thelevel of PDP-cysteine reagent incorporation on DNA was estimated fromthe optical adsorption ratio of pyridine-2-thione (λmax 343 nm andextinction coefficient E=8.08×10³) and DNA (λmax 260 nm and extinctionE=6.6×10³) after treatment of 15 μg of the modified DNA with 5 mMdithiothreitol (Sigma Chemical Co.) for 1.5 h at 20° C.

[0183] The labeled DNA was treated with 20 mM dithiothreitol (DTT, SigmaChemical Co.) for 1 hour at 4° C. to generate free thiols on the labeledplasmid. Reverse micelles were prepared by dissolving 82 μL of 1 μg/μLCys-DNA in 2.2 mL C₁₂E₄/TMP (Wo=6.58). The mixtures were agitated usinga vortex stirrer until a transparent solution was obtained (usually 2min). After formation of the micelles, sodium periodate was added to afinal concentration of 2 mM with respect to the total aqueous portion tooxidize the thiols to disulfides. The samples were centrifuged for 1 minat 14,000 rpm to remove any aggregates. A control reaction was preparedfollowing the same procedure using non-labeled DNA. The samples wereincubated at 4° C. for 2 hours. The reverse micelle system was disruptedwith the addition of 55 μL ethanol, 275 μL of 20 mM MOPS pH 7.5, 0.1 mMEDTA buffer, and 1.1 mL ethyl acetate. The reaction was vortexed andseparated into two layers via centrifugation. The aqueous layer waswashed twice with 2 mL ethyl acetate and once with 3 mL diethyl ether.The samples were then analysed by agarose gel electrophoresis.

[0184] Results

[0185] Agarose gel electrophoresis, indicated that periodate oxidized,cysteine DNA was found to remain in the well (indicating intramolecularoxidation of cysteine groups on the DNA). The non-oxidized cysteine DNAmigrated into the gel similarly to the unmodified DNA control.

Example 14 Mouse Tail Vein Injections of Oxidized Cysteine-pDNA(pCI Luc)Complexes Formed in a Reverse Micelle

[0186] Procedure

[0187] PCILuc DNA (pDNA) (Zhang, G., Vargo, D., Budker, V., Armstrong,N., Knechtle, S., Wolff, J. A. Human Gene Therapy, 8, 1763-1772, 1997)was modified with Cysteine Label IT® at weight ratios of 0.1:1 and 0.2:1(reagent:DNA) at 37° C. for 1 hour. The labeled DNA was treated with 20mM dithiothreitol (DTT, Sigma Chemical Co.) for 1 hour at 4° C. togenerate free thiols on the labeled plasmid. Reverse micelles wereprepared as described in Example 13. For each weight ratio, both anoxidized (sodium periodate added to the reverse micelle) and anon-oxidized sample (no sodium periodate was added) were prepared. ThepDNA was isolated as previously described.

[0188] Five complexes were prepared as follows

[0189] Complex I: pDNA (pCI Luc, 30 μg) in 7.5 mL Ringers.

[0190] Complex II: 0.1:1 cysteine labeled pDNA (pCI Luc, 30 μg)non-oxidized, in 7.5 mL Ringers.

[0191] Complex III: 0.1:1 cysteine labeled pDNA (pCI Luc, 30 μg)oxidized in the reverse micelle,in 7.5 mL Ringers.

[0192] Complex IV: 0.2:1 cysteine labeled pDNA (pCI Luc, 30 μg)non-oxidized, in 7.5 mL Ringers.

[0193] Complex V: 0.2:1 cysteine labeled pDNA (pCI Luc, 30 μg) oxidizedin the reverse micelle, in 7.5 mL Ringers.

[0194] Plasmid delivery in the tail vein of ICR mice (n=3) was performedas described. Tail vein injections of 2.5 mL of the complex werepreformed using a 30 gauge, 0.5 inch needle.

[0195] One day after injection, the animal was sacrificed, and aluciferase assay was conducted on the liver. Luciferase expression wasdetermined as previously reported (Wolff, J. A., Malone, R. W.,Williams, P., Chong, W., Acsadi, G., Jani, A. and Felgner, P. L. Directgene transfer into mouse muscle in vivo. Science, 1465-1468,1990.). ALumat LB 9507 (EG&G Berthold, Bad-Wildbad, Germany) luminometer wasused.

[0196] Results 2.5 mL injections

[0197] Complex I: 17,113,000 RLU

[0198] Complex II: 21,111,000 RLU

[0199] Complex III: 11,998,000 RLU

[0200] Complex IV: 2,498,000 RLU

[0201] Complex V: 4,498,000 RLU

[0202] Results

[0203] The luciferase assay indicates that the pDNA that is oxidizedwithin the reverse micelle is functional and able to be expressed.

Experiment 15. Synthesis of β-D-Glucopyranosyl Decane Disulfide andO-Glycine-β-D-Glucopyranosyl Decane Disulfide.

[0204]

[0205] Procedure

[0206] To a solution of decane thiol (0.59 mL, 2.9 mmol, AldrichChemical Company) in 11 mL CHCl₃ was added sulfuryl chloride (0.46 mL,5.7 mmol, Aldrich Chemical Company), and the resulting mixture wasstirred at room temperature for 18 h. Removal of solvent (aspirator),afforded decansulfenyl chloride.

[0207] To a solution of decansulfenyl chloride (190 mg, 0.92 mmol) in 4mL acetonitrile was added 1 -thio-β-D-glucose sodium salt hydrate (200mg, 0.92 mmol, Aldrich Chemical Company) and 15-crown-5 (0.18 mL, 0.899mmol, Aldrich Chemical Company). The resulting mixture was stirred atambient temperature for 16 h, filtered, and precipitated in Et₂O. Theresidue was triturated with Et₂O and purified by reverse phase HPLC onan Aquasil C18 column (Keystone Scientific Inc.), 10-90% B, 20 min (A=0.1% TFA in H₂O, B=0.1% TFA in Acetonitrile). Lyophilization afforded10 mg (3%) of β-D-glucopyranosyl decane disulfide as a fine white solid.

[0208] To a solution of β-D-glucopyranosyl decane disulfide (8 mg, 0.02mmol) in 80 μL THF was added N-Boc glycine (15 mg, 0.09 mmol, SigmaChemical Company), DCC (18 mg, 0.09 mmol, Aldrich Chemical Company), anda catalytic amount of dimethylaminopyridine (Aldrich Chemical Company).The resulting solution was stirred at ambient temperature for 12 h, andcentrifugated to remove the solid. The resulting solution wasconcentrated under reduced pressure, resuspended in dichloromethane,filtered through a plug of silica gel, and concentrated (aspirator). TheBoc protecting group was removed by taking the residue up in 200 μL of2.5% TIS / 50% TFA / dichloromethane for 12 h. Removal of solvent(aspirator), followed by purification by reverse phase HPLC on a AquasilC18 column (Keystone Scientific Inc.), 10-90% B, 20 min (A =0.1% TFA inH₂O, B=0.1% TFA in Acetonitrile) afforded 0.7 mg (5%) ofO-glycine-β-D-glucopyranosyl decane disulfide as a fine white solidfollowing lyophilization.

Example 16 Synthesis of β-D-Glucopyranosyl Cholesterol Disulfide

[0209]

[0210] By similar methodology as described in example 15,β-D-glucopyranosyl cholesterol disulfide was isolated (12% yield).

Experiment 17 Synthesis of Two Tailed β-D-Glucopyranosyl DisulfideDerivatives β-D-Glucopyranosyl N-Dodecanoyl-Cysteine-DodecanoateDisulfide and O-Glycine-β-D-GlucopyranosylN-Dodecanoyl-Cysteine-Dodecanoate Disulfide

[0211]

[0212] Procedure

[0213] To a solution of N-FMOC-S-Trt-Cysteine (585 mg, 1.0 mmol,NovaBioChem) in 4 mL dichloromethane was added 1-dodecanol (240 mg, 1.3mmol, Aldrich Chemical Company), DCC (260 mg, 1.3 mmol, Aldrich ChemicalCompany), and a catalytic amount of dimethylaminopyridine (AldrichChemical Company). The resulting solution was stirred at ambienttemperature for 30 min, filtered, and purified by flash chromatographyon silica gel (10-20% EtOAc/hexane eluent). Removal of solvent(aspirator) afforded 572 mg (76%) of the protected cysteine-dodecanoate.

[0214] To a solution of protected cysteine-dodecanoate (572 mg, 0.76mmol) was added 3 mL of 20% piperidine in DMF. The resulting solutionwas stirred at ambient temperature for 1 h, and partitioned inEtOAc/H20. The aqueous layer was extracted 2× EtOAc. The combinedorganic layer was washed 2× IN HCl, dried (Na2SO4), and concentrated toafford S-Trt-cysteine-dodecanoate. The residue was suspended in 2 mLdichloromethane, and cooled to −20° C. Diisopropylethylamine (0.16 mL,0.92 mmol, Aldrich Chemical Company) was added followed dodecanoylchloride (0.26 mL, 1.1 mmol, Aldrich Chemical Company), and the solutionwas allowed to slowly warm to ambient temperature. After 1 h, thesolvent was removed (aspirator), and the residue partitioned inEtOAc/H2O. The organic layer was washed 2×1 N HCl, 1×brine, dried(Na2SO4), and the solvent was removed (aspirator). The resulting residuewas suspended in 2% TIS / 50% TFA/dichloromethane to remove the tritylprotecting group. After 4 h the solution was concentrated, and theresulting residue was purified by flash column chromatography on silicagel (10-20% EtOAc/hexanes eluent) to afford 180 mg (42%)N-dodecanoyl-cysteine-dodecanoate (M+1=472.6).

[0215] To a solution of N-dodecanoyl-cysteine-dodecanoate (180 mg, 0.38mmol) in 0.5 mL chloroform was added sulfuryl chloride (62 L, 0.76 mmol,Aldrich Chemical Company). The resulting solution was stirred at ambienttemperature for 2 h and the solvent was removed (aspirator). Theresulting residue was suspended in 1 mL acetonitrile, and1-thio-β-D-glucose sodium salt hydrate (85 mg, 0.39 mmol, AldrichChemical Company) and 15-crown-5 (76 L, 0.38 mmol, Aldrich ChemicalCompany) were added. After 1 h at ambient temperature the solvent wasremoved (aspirator) and the residue was partitioned in EtOAc/H2O. Theorganic layer was concentrated and the resulting residue was purified byflash column chromatography on silica gel (5-10% MeOH/0.1% TFA /dichloromethane eluent) to afford 19 mg (8%) β-D-glucopyranosylN-dodecanoyl-cysteine-dodecanoate disulfide.

[0216] To a solution of β-D-glucopyranosylN-dodecanoyl-cysteine-dodecanoate disulfide (3.9 mg, 0.0045 mmol) in 100L dichloromethane was added N-Boc glycine (3.2 mg, 0.018 mmol, SigmaChemical Company), DCC (3.8 mg, 0.018 mmol, Aldrich Chemical Company),and a catalytic amount of dimethylaminopyridine (Aldrich ChemicalCompany). The resulting solution was stirred at ambient temperature for4 h, and filtered. The Boc protecting group was removed by taking theresidue up in 2 mL of 1% TIS/50% TFA/dichloromethane for 2 h. Removal ofsolvent (aspirator), followed by purification by reverse phase HPLC on aDiphenyl column (Vydaq), 20-90% B, 20 min (A=0.1% TFA in H₂O, B=0.1% TFAin Acetonitrile) afforded 3.6 mg (90%) of O-glycine-β-D-glucopyranosyldecane disulfide as a fine white solid following lyophilization.

Experiment 18 Synthesis of Disulfide Containing Surfactants 1).Synthesis of the Disulfide of Decanethiol and 3-Dimethylamino-Thiopropionamide

[0217]

[0218] Procedure

[0219] To a solution of thiopropionic acid (0.41 mL, 4.7 mmol, AldrichChemical Company) in 18 mL CH₂Cl₂ was added diisopropylethylamine (0.82mL, 4.7 mmol, Aldrich Chemical Company) followed by trityl chloride (1.4g, 4.9 mmol, Aldrich Chemical Company). The resulting mixture wasstirred at room temperature for 18 h. Removal of solvent (aspirator)afforded a white crystalline solid. The material was partitioned inEtOAc/H₂O, and washed with 0.1 M NaHCO₃ and 1×brine. Concentrated toafford S-trityl thiopropionic acid.

[0220] To a solution of S-trityl-thiopropionic acid (0.30 g, 0.86 mmol)in 3.5 mL CH₂Cl₂ was added PyBOP (0.45 g, 0.86 mmol, NovaBioChem). Themixture was stirred at ambient temperature for 5 min and thendimethylaminopropylamine (0.11 mL, 0.86 mmol, Aldrich Chemical Company)was added. The solution was stirred at room temperature for 18 h, andconcentrated. The residue was brought up in EtOAc and partitioned inH₂O. The organic layer was washed 2×H₂O, 1×brine, dried (Na₂SO₄), andthe solvent removed (aspirator). The resulting residue was suspended in2% TIS/50% TFA/ CH₂Cl₂ (3 mL) to remove the trityl protecting group.After 2 h the solution was concentrated to afford 3-dimethylamino-thiopropionamide.

[0221] To a solution of 3-dimethylamino- thiopropionamide (0.082 g, 0.43mmol) in 1.5 mL dichloromethane was added decanethiolchloride (0.090 g,0.43 mmol, prepared as in example 15). The resulting solution wasstirred at ambient temperature for 20 min. The solvent was removed andthe resulting residue was purified by flash column chromatography onsilica gel (15% MeOH/CH₂Cl₂ eluent) to afford 17.2 mg (9%) of thedisulfide of decanethiol and 3-dimethylamino-thiopropionamide(M+1=363.4).

2). Synthesis of the Disulfide of Dodecanethiol and3-Dimethylamino-Thiopropionamide

[0222]

[0223] Procedure

[0224] By a similar procedure as above,thiopropyl-dimethylaminopropylamine (0.10 g, 0.52 mmol) in 2.0 mLdichloromethane was added dodecanethiolchloride (0.12 g, 0.52 mmol). Theresulting solution was stirred at ambient temperature for 20 min. Thesolvent was removed and a portion of the resulting residue (160 mg) waspurified by flash column chromatography on silica gel (10% MeOH/CH₂Cl₂eluent) to afford 22.4 mg (14%) of the disulfide of dodecanethiol and3-dimethylamino- thiopropionamide (M+1=391.4).

3). Synthesis of the Disulfide of Decanethiol andThiopropionic-3-Dimethylaminopropanoate

[0225]

[0226] Procedure

[0227] To a solution of trityl-S-thiopropionic acid (0.36 g, 1.0 mmol)in 4.0 mL CH₂Cl₂ was added PyBOP (0.54 g, 1.0 mmol, NovaBioChem). Themixture was stirred at ambient temperature for 5 min before the additionof dimethylaminopropanol (0.12 mL, 1.0 mmol, Aldrich Chemical Company).The solution was stirred at room temperature for 18 h, and concentrated.The residue was brought up in EtOAc and partitioned in H₂O. The organiclayer was washed 2×H₂O,1×brine, dried (Na₂SO₄), and the solvent removed(aspirator). The resulting residue was suspended in 2% TIS/50%TFA/CH₂Cl₂ (3 mL) to remove the trityl protecting group. After 2 h thesolution was concentrated to affordthiopopionic-3-dimethylaminopropanoate.

[0228] To a solution of thiopopionic-3-dimethylaminopropanoate (0.10 g,0.52 mmol) in 2 mL dichloromethane was added decanethiolchloride (0.11g, 0.52 mmol). The resulting solution was stirred at ambient temperaturefor 20 min. The solvent was removed and a portion of the resultingresidue (25 mg) was purified by plug filtration on silica gel (10%MeOH/CH₂Cl₂ eluent) to afford 20.9 mg (84%) of the disulfide ofdecanethiol and thiopopionic-3-dimethylaminopropanoate (M+1=364.4).

4). Synthesis of the Disulfide of Dodecanethiol andThiopopionic-3-Dimethylaminopropanoate

[0229]

[0230] Procedure

[0231] To a solution of thiopopionic-3-dimethylaminopropanoate (0.10 g,0.52 mmol) in 2 mL dichloromethane was added dodecanethiolchloride (0.11g, 0.52 mmol). The resulting solution was stirred at ambient temperaturefor 20 min. The solvent was removed and a portion of the resultingresidue (150 mg) was purified by flash column chromatography on silicagel (1% TFA/10% MeOH/CH₂Cl₂ eluent) to afford 38 mg (25%) of thedisulfide of decanethiol and thiopopionic-3-dimethylaminopropanoate(M+1=392.4).

Experiment 19 Synthesis of Silicone Containing Amphipathic Molecules 1.Synthesis of 3-dimethylamino-dimethyloctadecyl Silyl Ether

[0232]

[0233] To a solution of 3-dimethylamino-1-propanol (0.873 mmol, AldrichChemical Company) in 2 mL chloroform was added dimethyloctadecylchlorosilane (378 mg, 1.09 mmol, Aldrich Chemical Company) and imidazole(74.2 mg, 1.09 mmol, Aldrich Chemical Company). After 16 hrs at ambienttemperature, the solution was partitioned in EtOAc/H2O with 10% sodiumbicarbinate. The organic layer was washed with water, and brine. Thesolvent was removed (aspirator) to afford 328 mg ( 91%) of3-dimethylamino-dimethyloctadecyl silyl ether as a cream colored solid.

2. Synthesis of 3-(dimethylamino)-1,2- dimethyloctadecyl Silyl Ether

[0234]

[0235] To a solution of 3-(dimethylamino)-l,2-propanediol (50.0 mg,0.419 mmol, Aldrich Chemical Company) in 2 mL chloroform was addeddimethyloctadecyl chlorosilane (328 mg, 0.944 mmol, Aldrich ChemicalCompany) and imidazole (68.1 mg, 0.944 mmol, Aldrich Chemical Company).After 16 hrs at ambient temperature, the solution was partitioned inEtOAc/H2O with 10% sodium bicarbinate. The organic layer was washed withwater, and brine. The solvent was removed (aspirator) to afford 266 mg(86%) of 3-(dimethylamino)-1,2- dimethyloctadecyl silyl ether as a whitesolid.

Experiment 20 Application of Reverse Micellar Formulations to MouseDermis

[0236] Procedure

[0237] Five Complexes were prepared:

[0238] Complex I. Doxorubicine hydrochloride was dissolved in water to afinal concentration of 5.8 mg/mL. To a solution of 12 L Brij 30 (SigmaChemical Company) in 88 L of tetramethylpentane was added 5 μL of thedoxorubicine hydrochloride solution. The sample was vortexed for 2 minresulting in a clear red solution.

[0239] Complex II Doxorubicine hydrochloride was dissolved in water to afinal concentration of 50 mg/mL. To a solution of 10 L of Brij 30 (SigmaChemical Company) and 2 mg β-D-glucopyranosyl decane disulfide in 190 Lof tetramethylpentane was added 5 μL of the doxorubicine hydrochloridesolution. The sample was vortexed for 2 min resulting in a clear redsolution.

[0240] Complex III Doxorubicine hydrochloride was dissolved in water toa final concentration of 50 mg/mL. To a solution of 10 L of Brij 30(Sigma Chemical Company) and 0.5 mg O-Glycine-β-D-glucopyranosyl decanedisulfide in 190 L of tetramethylpentane was added 5 μL of thedoxorubicine hydrochloride solution. The sample was vortexed for 2 minresulting in a clear red solution.

[0241] Complex IV Doxorubicine hydrochloride was dissolved in water to afinal concentration of 50 mg/mL. To a solution of 10 L of Brij 30 (SigmaChemical Company) and 6 mg 3-dimethylamino-dimethyloctadecyl silyl etherin 190 L of tetramethylpentane was added 5 μL of the doxorubicinehydrochloride solution. The sample was vortexed for 2 min resulting in aclear red solution.

[0242] Complex V Doxorubicine hydrochloride was dissolved in water to afinal concentration of 50 mg/mL. To 200 L H₂O was added 5 μL of thedoxorubicine hydrochloride solution.

[0243] ICR mice were anesthetize, and the hair removed from the back ofthe neck, and on one animal the abdominal skin. After 1 h the animalswere sacrificed, and the skin samples removed and examined. Thecomplexes were applied to the dermis as follows: Complex I. The complexwas applied by immersing a cotton swap in the solution, and swabbing theabdominal skin and the dehaired skin on the back of the neck.

[0244] Complex II-V. The complex was applied by dropping 50 L ofsolution onto the back of the neck.

[0245] Results

[0246] Fluorescent examination of the skin samples (O.C.T. frozen, UVlight). Samples from the application of Complex I were showed a muchlower level of positive cells than from Complexes II-IV. Complex NumberLocation of the label I Abdominal Positive label is restricted to nucleionly with skin majority of them being epithelium cells. Small portion ofpositive sells are connective tissue cells adjoining to the labeledepithelium cells. Skin from Similar pattern of labeling. the back II7477 Whole epithelium compartment is very bright, not specificallynuclei. Some connective tissue cells in deeper part of derma arepositive. No positive follicular cells. 7479 Whole epitheliumcompartment is very bright, not specifically nuclei. Some connectivetissue cells in deeper part of derma are positive. Very rare positivefollicular cells. III 6939 Whole epithelium compartment is very bright,not specifically nuclei. Some follicular cells are positive. 7459 Wholeepithelium compartment is very bright, not specifically nuclei. Somefollicular cells are positive IV 7476 Whole epithelium compartment ispositive but less than in previous two groups, some connec- tive tissuecells in deeper part of derma are positive. 7460 Whole epitheliumcompartment is positive, some connective tissue cells in deeper part ofderma are positive. V 7474 Mostly only the skin surface is positive,occasionally some deeper cells, probably damaged areas (shaving) Cellsand nuclei are negative. 7463 Mostly only the skin surface is positive,occasionally. Cells and nuclei are negative. Water Negative

[0247] Analysis

[0248] Reverse micelles are to incorporate doxorubicine hydrochlorideand deliver the drug to the epithelium.

[0249] New Definitions to Include in the Specification

[0250] A reactive functional group means a bond that can undergochemical modification or reaction.

[0251] The term cargo means pharmaceuticals, proteins, peptides,hormones, cytokines, antigens and small molecules.

[0252] Substructure means the chemical structure of the compound and anycompounds derived from that chemical structure from the replacement ofone or more hydrogen atoms by any other atom or change in oxidationstate. For example if the substructure is succinic anhydride, thenmethylsuccinic anhydride, 2,2-dimethylsuccinic anhydride,3-oxabicyclo[3.1.0]hexane-2,4-dione, maleic anhydride, citriconicanhydride, and 2,3-dimethylmaleic anhydride have the same substructure.

[0253] The foregoing examples are considered as illustrative only of theprinciples of the invention. Further, since numerous modifications andchanges will readily occur to those skilled in the art, it is notdesired to limit the invention to the exact construction and operationshown and described. Therefore, all suitable modifications andequivalents fall within the scope of the invention.

We claim:
 1. A process for forming a negatively-charged, zwitterionic,or neutral complex for delivery to a cell, comprising: a) forming acationic reverse micelle using amphipathic molecules, b) inserting abiologically active compound into the cationic reverse micelle, c) thenchanging the charge of the cationic reverse micelle to anegatively-charged, zwitterionic, or neutral reverse micelle anddelivering it to the cell.
 2. The process of claim 1 wherein theamphipathic molecule contains a reactive functional group.
 3. Theprocess of claim 2 wherein the reactive functional group consists of agroup capable of participating in a polymerization reaction.
 4. Theprocess of claim 1 wherein the amphipathic molecule contains a labilebond.
 5. The process of claim 4 wherein the labile bond consists of adisulfide bond.
 6. The process of claim 5 wherein the amphipathicmolecule contains a reactive functional group.
 7. The process of claim 6wherein the reactive functional group consists of a group capable ofparticipating in a polymerization reaction.
 8. The process of claim 4wherein the amphipathic molecule contains a silicon-heteroatom bond. 9.The process of claim 8 wherein the amphipathic molecule contains areactive functional group.
 10. The process of claim 9 wherein thereactive functional group consists of a group capable of participatingin a polymerization reaction.
 11. The process of claim 4 wherein theamphipathic molecule contains an amide constructed from a compoundhaving a substructure of succinic anhydride.
 12. The process of claim 11wherein the amphipathic molecule contains a reactive functional group.13. The process of claim 12 wherein the reactive functional groupconsists of a group capable of participating in a polymerizationreaction.
 14. A process for forming a complex that is deliverable to acell, comprising: inserting a cargo into a reversemicelle consisting ofone or more amphipathic molecules wherein at least one of theamphipathic molecules contains a labile bond.
 15. The process of claim14 wherein the amphipathic molecule contains a reactive functionalgroup.
 16. The process of claim 15 wherein the reactive functional groupconsists of a group capable of participating in a polymerizationreaction.
 17. The process of claim 14 wherein the amphipathic moleculecontains a disulfide bond.
 18. The process of claim 17 wherein theamphipathic molecule contains a reactive functional group.
 19. Theprocess of claim 18 wherein the reactive functional group consists of agroup capable of participating in a polymerization reaction.
 20. Theprocess of claim 14 wherein the amphipathic molecule contains asilicon-heteroatom bond.
 21. The process of claim 20 wherein theamphipathic molecule contains a reactive functional group.
 22. Theprocess of claim 21 wherein the reactive functional group consists of agroup capable of participating in a polymerization reaction.
 23. Theprocess of claim 14 wherein the amphipathic molecule contains an amideconstructed from the compound having a substructure of succinicanhydride.
 24. The process of claim 23 wherein the amphipathic moleculecontains a reactive functional group.
 25. The process of claim 24wherein the reactive functional group consists of a group capable ofparticipating in a polymerization reaction.
 26. A negatively-charged,zwitterionic, or neutral compound which is deliverable to a mammaliancell, comprising: a negatively-charged, zwitterionic, or neutral micellecontaining a biologically active molecule.